| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
First, in order to advance the various researches in laboratory animals, we examined fundamental measurement conditions for detecting reactive oxygen species of rat peripheral neutrophils by use of flow cytometry. For measuring phagocytic activity with flow cytometry, Staphylococcus aureus was labeled with fluoresce in isothiocynate (FITC) and used as phagocytic particles. We decided the optimal concentrations of FITC in regard to the fluorescent intensity. As for the measurement of reactive oxygen-producing activity, hydroethidine (HE) which reacts with superoxide was used and the suitable concentration was determined by dependency of the fluorescent intensity. Furthermore, simultaneous measurement of both of these activities was made possible by detecting FITC and HE at the different wave length. In addition, nitroblue tetrazolium (NBT) reduction method was also performed using the same samples. As a result, strong correlations were confirmed between both measurement methods.
Next, we
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investigated the fundamental measurement conditions in case of human peripheral blood. It was found that hemolysis procedure after the cellular reaction could affect flow cytometry distribution of neutrophils, lymphocytes and monocytes, and we improved the operation methods. On the other hand, isolated neutrophils lose activity soon, and there was a necessity to measure reactive oxygen production soon after the separation from the whole blood. Then we tried to examine the high sensitivity detection probe by comparing HE with Dihydrorhodamine (DHR) -123, Dichrolo Fluorescein Diacetate (DCFH-DA). DHR-123 was superior in sensitivity, by coexisting NaN3 which is the inhibitor of myeloperoxidase, compared with DCFH-DA. Concerning the phagocytic particles, FITC-labeled Staphylococcus aureus and zymosan were used, but the culture of pathogens is dangerous and not easy to handle to label with FITC. In addition, zymosan caused aggregation of neutrophils and was not suitable for the measurement of flow cytometry method. For those reasons, Carboxylate Microspheres of the diameter 1 μ m with fluorescence dye was used, and it became possible to measure phagocytic activity without such difficulties. We could set up the detection wave length of Carboxylate Microspheres without interference of those of HE and DHR-123, and simultaneous analyses of phagocytosis and reactive oxygen production became possible.
Furthermore, by applying the principle of flow cytometry that can measure several indices simultaneously by changing detection wave length, the simultaneous measurement of various molecules using human plasma samples became possible. Conventional enzyme-linked immunosorbent assay (ELISA) takes larger sample volume and takes time for the measurement. On the other hand, this method have greater range of measurement, but the sensitivity was poorer than ELISA method. Taken together, flow cytometry method is enable us to measure multi-channel parameters that we could not do until recently. Less
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